Reference sequence statistics midway.

The HIV-1 provirus mainly consists of internal coding region flanked by 1 long terminal repeats (LTRs) at each terminus. The LTRs play important roles in HIV-1 reverse transcription, integration, and transcription. However, despite of the significant study advances of the internal coding regions of HIV-1 by using definite reference classification, there are no systematic and phylogenetic classifications for HIV-1 5’ LTRs, which hinders our elaboration on 5’ LTR and a better understanding of the viral origin, spread and therapy. Here, by analyzing all available resources of 5’ LTR sequences in public databases following 4 recognized principles for the reference classification, 83 representatives and 14 consensus sequences were identified as representatives of 2 groups, 6 subtypes, 6 sub-subtypes, and 9 CRFs. To test the reliability of the supplemented classification system, the constructed references were applied to identify the 5’ LTR assignment of the 22 clinical isolates in China. The results revealed that 16 out of 22 tested strains showed a consistent subtype classification with the previous LTR-independent classification system. However, 6 strains, for which recombination events within 5’ LTR were demonstrated, unexpectedly showed a different subtype classification, leading a significant change of binding sites for important transcription factors including SP1, p53, and NF-κB. The binding change of these transcriptional factors would probably affect the transcriptional activity of 5’ LTR. This study supplemented a unified classification system for HIV-1 5’ LTRs, which will facilitate HIV-1 characterization and be helpful for both basic and clinical research fields.HIV-1前病毒（HIV-1 provirus）主要由内部编码区构成，该编码区的两端各带有1个长末端重复序列（long terminal repeats, LTRs）作为侧翼序列。LTRs在HIV-1逆转录、整合及转录过程中发挥关键作用。尽管学界已通过规范的参考分类方法，在HIV-1内部编码区的研究上取得了显著进展，但目前尚无针对HIV-1 5’端LTR的系统性发育进化分类体系，这一现状阻碍了我们对5’LTR的深入阐释，也制约了对病毒起源、传播与治疗的更全面认知。本研究遵循4项公认的参考分类原则，对公共数据库中所有可获取的5’LTR序列资源进行分析，最终鉴定出83条代表性序列与14条共有序列，可归为2个组、6个亚型、6个亚亚型以及9个循环重组型（CRFs）。为验证所补充构建的分类体系的可靠性，研究人员将所构建的参考序列集用于鉴定中国境内22株临床分离株的5’LTR分型，结果显示，22株受试毒株中有16株的亚型分类结果与此前不依赖LTR的分类体系一致。然而，另有6株经证实存在5’LTR内重组事件的毒株，其分型结果却出乎意料地出现偏差，导致包括SP1、p53以及NF-κB在内的重要转录因子结合位点发生显著改变，这类转录因子结合位点的变化可能会影响5’LTR的转录活性。本研究补充构建了一套针对HIV-1 5’LTR的统一分类体系，该体系将有助于推动HIV-1的特征鉴定研究，并为基础与临床相关研究领域提供有力支撑。